Results Selection for virus-sensitive clones from R4-7 mutant cells expressing cDNAs The R4-7 mutant cell line is approximately 100-fold resist-ant to transduction by MuLV-based vectors
Trang 1Open Access
Research
Isolation of suppressor genes that restore retrovirus susceptibility
to a virus-resistant cell line
Guangxia Gao1,2 and Stephen P Goff*1
Address: 1 Department of Biochemistry and Molecular Biophysics Howard Hughes Medical Institute Columbia University College of Physicians and Surgeons New York NY 10032, USA and 2 Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China
Email: Guangxia Gao - gaogx@sun.im.ac.cn; Stephen P Goff* - goff@cancercenter.columbia.edu
* Corresponding author
Abstract
Background: Genetic selections in mammalian cell lines have recently been developed for the
isolation of mutant cells that are refractory to infection by retroviruses These selections have been
used to recover lines that block early postentry stages of infection, either before reverse
transcription or before nuclear entry The mechanisms of action of these blocks remain unknown
Results: We have devised a method for the selection of genes from cDNA libraries that suppress
the block to virus infection, and so restore virus susceptibility The protocol involves the
transformation of pools of resistant cells by cDNA expression libraries, followed by the selection
for rare virus-sensitive cells, using multiple rounds of selection after infection by marked viral
vector genomes The suppressor genes were then recovered from these virus sensitive cells, and
their ability to restore virus susceptibility was confirmed by reintroduction of these cDNAs into
the resistant line
Conclusions: The identities of these genes provide insights into the mechanism of virus resistance
and will help to define new pathways used during retrovirus infection The methods for gene
isolation developed here will also permit the identification of similar suppressors that modify or
override other recently identified virus resistance genes
Background
It is becoming increasingly apparent that mammalian
cells harbor numerous genes that induce intracellular
blocks to retrovirus infection [1,2] These genes have
pre-sumably evolved and been maintained in the genome in
response to the pathogenic and lethal consequences of
infection, and are now thought to constitute an important
part of the host defense against these viruses Some of the
genes and gene products responsible for this resistance
have been recently identified, including the Fv1 locus in
the mouse, which blocks infection after reverse
transcrip-tion but before nuclear entry and establishment of the
integrated provirus [3]; the APOBEC3G enzyme, which is incorporated into virion particles and catalyzes the destructive deamination of the viral cDNA during reverse transcription [4]; and the TRIM5a protein, which some-how blocks incoming virus soon after entry and prevents the activation of reverse transcription [5] Others likely remain to be identified
We have been involved in the development of screens and selections for virus resistance genes, and have isolated mutant cell lines after chemical mutagenesis that are pro-foundly resistant to retrovirus infection Two such lines
Published: 28 September 2004
Retrovirology 2004, 1:30 doi:10.1186/1742-4690-1-30
Received: 24 August 2004 Accepted: 28 September 2004
This article is available from: http://www.retrovirology.com/content/1/1/30
© 2004 Gao and Goff; licensee BioMed Central Ltd
This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Trang 2isolated from a parental fibroblast cell line, Rat2 cells,
have been characterized in some detail [6] Mutant line
R3-2 exhibited a nearly 1000-fold resistance to infection
by genetically marked Moloney murine leukemia virus
genomes, and was resistant to pseudotyped viruses
utiliz-ing the ecotropic envelope, the amphotropic envelope, or
even the VSV G envelope protein Infection of R3-2
resulted in the normal synthesis of the linear viral DNA by
reverse transcription, but circular viral DNAs and
inte-grated proviruses were not generated The viral DNA was
apparently trapped in the cytoplasm in a form that was
not readily extracted by conditions that allowed DNA
recovery from wild-type infected cells Mutant line R4-7
exhibited about a 100-fold resistance to infection by
M-MuLV, also independent of the envelope mediating entry
Infection of this line was blocked earlier, before the
initi-ation of reverse transcription Both lines R3-2 and R4-7
were also resistant to infection by pseudotyped HIV-1
viral vectors
To probe the nature of the blocks in these mutant cell
lines, we have sought to identify and characterize
suppres-sor genes that override the restriction exhibited by these
cells To identify such genes, we have developed
method-ologies that allow for the selection of rare virus-sensitive
clones arising after transfer of gene libraries into
popula-tions of virus-resistant parents We here report the
isola-tion of two cDNA constructs that each restore virus
sensitivity to the R4-7 mutant cell line These DNAs
con-stitute valuable tools in the characterization of this line's
virus resistance
Results
Selection for virus-sensitive clones from R4-7 mutant cells
expressing cDNAs
The R4-7 mutant cell line is approximately 100-fold
resist-ant to transduction by MuLV-based vectors as compared
to wild-type Rat2 cells [6] To identify genes that could
suppress this phenotype and restore virus sensitivity, a
protocol involving multiple rounds of selection for virus
sensitivity was devised (Fig 1) First, R4-7 cells were
trans-formed by a library of rat kidney cDNAs expressed from
the constitutive CMV promoter Recipient cells were
selected by cotransformation with a DNA expressing
puromycin resistance Five pools of the
puromycin-resist-ant cells were generated and maintained separately, each
pool containing more than 1000 independent
trans-formed clones The expectation was that multiple rounds
of selection for virus-sensitive clones would be required to
recover such cells, with each round providing at most a
100-fold enrichment
Four of the pools of transformed cells, with each clone in
the pools overexpressing a small number of cDNAs, were
sequentially exposed to a series of three genetically
marked ecotropic MuLV-based vectors, and the rare suc-cessfully infected cells were recovered after each infection
by selection for the marker carried by the vector (see Methods) The cells were first exposed to N2 virus, an MuLV vector carrying the neor marker, and infected cells were selected in medium containing G418 These cultures were then expanded and exposed to Eco-TK virus, an MuLV vector carrying the Herpes virus TK gene, and infected cells were selected with HAT medium These cul-tures were expanded and finally exposed to Eco-His virus, and infected cells were selected with medium containing histidinol In all cases, the selecting viral vectors were applied at low multiplicities of infection (MOI) so as not
to override the resistance of the parental R4-7 cells, as can happen at high MOI [6] Individual colonies were recov-ered after the triple selection
The number of colonies of infected cells recovered at each stage of the selection was determined for each of the four pools (Table 1) The number of colonies of wild-type Rat2 cells exposed to the virus in parallel was determined for comparison In each of the first two rounds of selection, the pools of mutant cells yielded about 25-fold fewer transductants than the wild-type control, indicating reten-tion of the resistance in the bulk of the populareten-tion In the third round, pools 3 and 4 yielded slightly higher num-bers of colonies than the other pools, suggesting possible enrichment for virus sensitive clones, though still less than the wild-type cells A total of 36 candidate colonies were isolated
To determine whether any of these candidate clones had become truly virus sensitive, all 36 colonies were individ-ually picked and expanded into larger cultures These cul-tures were then tested by infection with Eco-GFP, a virus vector expressing the green fluorescent protein, and the fraction of the cells expressing the marker was determined
by inspection While all the clones from pools 1 and 2 were as resistant as the parental R4-7 line, a total of 6 clones – 2 from pool 3 (dubbed A1, A2) and 4 from pool
4 (dubbed B1, B2, C1, C2) – were fully sensitive to infec-tion These cloned lines were thus candidates as poten-tially carrying cDNAs that could restore virus sensitivity to the R4-7 line
Recovery of cDNAs from virus-sensitive cell lines capable
of suppressing virus resistance
To recover the cDNAs present in the virus sensitive cell lines, total genomic DNA was isolated, and polymerase chain reactions were performed to amplify expression cas-settes composed of the CMV promoter, the cDNA insert of the library and the poly(A) addition signal The amplified DNA from each line was directly cloned into the TOPO plasmid DNA and used to transform bacteria In this way cloned cDNAs were recovered from five of the six lines
Trang 3Flowchart for isolation of cDNAs that suppress virus resistance and restore virus sensitivity to the R4-7 mutant cell line
Figure 1
Flowchart for isolation of cDNAs that suppress virus resistance and restore virus sensitivity to the R4-7 mutant cell line See text for description
Schematic of protocol for isolation of suppressor cDNAs
Cotransformation, puromycin selection
cDNA expression library
pGK-puro
Transformed pools
+
Infection, G418 selection
N2 Virus
Infection, HAT selection
TK Virus
Infection, Histidinol selection
His Virus
Transduced cells
Transduced cells
Transduced cells
Pick colonies
GFP Virus
Candidate
Test each line for virus susceptibility
Recover cDNA inserts
Test DNAs for suppressor activity in R4-7
Active Suppressor cDNAs
Candidate cDNAs
Trang 4Because the lines were expected to each carry a few
differ-ent cDNAs, and because only one cDNA in each line
would be expected to be responsible for the phenotype, a
total of 50 bacterial colonies were isolated for each of the
five lines DNAs were prepared from these bacterial
colo-nies and assigned to groups based on the pattern of
restric-tion fragments after produced after digesrestric-tion with MspI
The number of distinct cDNAs recovered from each of the
five lines ranged from 1 to 11, and all together included
29 cDNAs (Table 2)
The cDNAs isolated from the virus sensitive lines were
then tested directly for their ability to suppress the virus
resistance of R4-7 cells Each cDNA (20 ug) was mixed
with pGK-puro DNA (2 ug) and used to transform naive
R4-7 cells, and recipients stably expressing the DNAs were
selected by growth in puromycin The resulting
puromy-cin-resistant colonies derived from a given cDNA were
pooled and grown into large cultures, and the resulting
populations were tested for sensitivity to Eco-neo virus
infection Two of the cDNAs, one from cell line B1
(desig-nated pB1-11) and one from cell line C1 (desig(desig-nated
pC1-2), dramatically suppressed the virus resistance of R4-7
cells (Fig 2) An inactive cDNA retained as a negative
con-trol did not suppress the resistance The susceptibility to
infection of the pooled R4-7 transfectants for the two
active clones was similar to that of the wild-type Rat2
cells, and roughly 100-fold higher than that of the R4-7
parents To further document the sensitivity of induced by
pC1-2, two individual clones were isolated from the R4-7
populations expressing pC1-2 and a control cDNA, and these clones were similarly tested by infection with Eco-neo virus Like the pooled populations, the clones expressing pC1-2 were virus-sensitive and the controls were not (Fig 3) Thus, these cDNAs were sufficient to suppress the resistance, and were likely responsible for the virus sensitivity of the two lines in which they were recov-ered after the triple selection The remaining lines had pre-sumably become sensitive to virus independently of any
of the cDNAs they carried, or as a result of a cDNA that was not recovered from the PCR amplified DNA products
Characterization of biologically active suppressor cDNAs
The pB1-11 and pC1-2 DNAs could function as general enhancers of retrovirus infection, or alternatively as specific suppressors of the block in the R4-7 mutant cell line To distinguish between these possibilities, the DNAs were introduced into the wild-type Rat2 cells, the distinct R3-2 mutant line, and the R4-7 line by cotransformation, and stable transformants were selected and expanded The resulting transformed lines were then tested for their sen-sitivity to infection by Eco-Neo virus The Rat 2 lines expressing pB1-11 showed no change in virus susceptibil-ity, and the Rat2 lines expressing pC1-2 showed at most a 2-fold increase in sensitivity (Fig 4) The corresponding R3-2 lines gave similar results (data not shown) Thus, both cDNAs were highly specific in enhancing the virus susceptibility of the R4-7 line
Table 1: Numbers of colonies recovered after each round of infection and selection
Initial Cell Population Pool 1 Pool 2 Pool 3 Pool 4 Rat2 Puro R colonies after transfection >1000 >1000 >1000 >1000 -Neo R colonies after N2 virus infection ~400 ~400 ~400 ~400 TMTC
-Table 2: Numbers of cDNAs recovered from Virus S cell lines
Pool 3 Pool 3 Pool 4 Pool 4 Pool 4 Pool 4
Trang 5The DNA sequences of the two cDNAs were determined and compared with the nucleic acid sequences of the NCBI databases Clone pB1-11 contained an insert of 855
bp with close sequence similarity to the central portion of
a transcript originally termed HCC1.3/1.4, identified as encoding a prominent autoantigen expressed in a human hepatocarcinoma [7] The similar mouse gene product, dubbed CAPER, was subsequently shown to interact with c-Jun, a subunit of the AP-1 activator, and the estrogen receptors ERa and ERß, and to exhibit transcriptional coactivator activity when expressed in concert with these transcription factors [8] The cDNA insert of pB1-11 aligned well with both the human sequences (92% iden-tity match to bp 910–1767 of HCC1.4 (Genbank acces-sion no L10911)) and the mouse sequences (94% identity match to bp 1153–2006 of CAPER (accession no AY061882)) Remarkably, the cDNA fragment was inserted in reverse orientation relative to the CMV pro-moter of the pcDNAI plasmid vector [9], and thus the active DNA would produce an antisense mRNA transcript Clone pC1-2 proved to contain an insert of 1407 bp, with close sequence similarity to a central portion of the VL30
Ability of the suppressor cDNAs to restore virus susceptibility to the R4-7 mutant cell line
Figure 2
Ability of the suppressor cDNAs to restore virus susceptibility to the R4-7 mutant cell line R4-7 cells were cotransformed with the indicated cDNAs, and the transformants were pooled and grown into cell populations These cultures were then exposed to equal amounts (approximately 10,000 cfu in NIH/3T3 cells) of an N2 virus preparation, and virus susceptibility was assessed by plating the infected cells in medium containing G418 While the mutant R4-7 control populations yielded only ~50 colonies, the populations expressing the active cDNAs produced nearly confluent lawns Rat2: virus-sensitive subclone isolated after mutagenesis R4-7: mutant line No cDNA: pGKpuro marker DNA alone Control cDNA: marker plus inactive cDNA PB1-11, pC1-2: marker plus indicated cDNA
G418 r colonies after N2 infection of R4-7 populations
expressing the indicated cDNA
Rat2
no cDNA
R4-7
no cDNA
R4-7 control cDNA
R4-7 pB1-11
R4-7 pC1-2
Restoration of virus susceptibility by pC1-2 DNA in clonal
cell lines
Figure 3
Restoration of virus susceptibility by pC1-2 DNA in clonal
cell lines Single-cell clones were derived from R4-7 cell
pop-ulations cotransformed with either inactive control cDNA or
pC1-2 DNA The resulting lines were exposed to N2 virus
(approximately 300 cfu in NIH/3T3 cells) and plated in
medium containing G418 While the control yielded no
colo-nies, the clonal lines containing pC1-2 showed 100–200
colonies
G418 r colonies after N2 virus infection of
R4-7 subclones expressing indicated cDNAs
Trang 6elements, a family of endogenous retrovirus-like elements
widely expressed in many mouse [10-14] and Rat cell
lines [15,16] The pC1-2 sequences aligned best with
par-ticular Rat elements expressed in tumor cells (~88%
iden-tity to bp 5025–6151 of a 7.4-kb element [17]; Genbank
accession no D90005) and in the ovary (~90% identity to
bp 3341–4677 of a 5.5-kb element [18]; Genbank acces-sion no U48828) There was weaker similarity to related retroviruses, such as the gibbon ape leukemia virus [19] The insert was in the sense orientation relative to the CMV promoter, and if transcribed would result in formation of
a plus strand RNA, corresponding to the central portion of the VL30 transcripts Like most rat VL30 elements, the insert did not include any significant open reading frames, but rather contained numerous mutations that intro-duced frameshifts and stop codons that would preclude synthesis of any long protein products These results sug-gest that both of the pB1-11 and pC1-2 DNAs might function by virtue of their RNA products rather than any encoded proteins The sequences of the two inserts have been submitted to the NCBI database (pB1-11 accession number is AY769432; pC 1-2 accession number is AY769433; see figure 5)
Expression of CAPER and VL30 RNAs in R4-7 mutant line
The biological activity of the pB1-11 and pC1-2 DNAs in restoring virus susceptibility could be mediated through effects on their corresponding endogenous gene products expressed in the R4-7 mutant cell line, or could be indi-rect If their activity was direct, then either one of the cor-responding endogenous genes – the CAPER gene or a VL30 element – might be the locus that was originally mutated to give rise to the resistance of the R4-7 line To examine this possibility, RNAs were prepared from R4-7 and wild-type cells, and analyzed by Northern blot Hybridizing with the pB1-11 probe showed a single major RNA about 3 kb in length in both lines, with no signifi-cant change in level detected (Fig 6) Hybridization with the pC1-2 probe showed an intense smear of RNAs in both lines as typically seen for VL30 RNAs (data not shown) No differences between the lines was apparent Although the levels of the CAPER mRNA was not detecta-bly altered in the R4-7 line, it remained possible that the gene and its transcripts harbored point mutations that were responsible for the virus resistance To test this pos-sibility, CAPER cDNAs were isolated from the R4-7 and Rat2 cells by RT-PCR, and the amplified sequences were cloned into the TOPO vector Ten cDNA clones from each line were recovered and sequenced Clones of three dis-tinct structures were recovered from each line, likely aris-ing by alternative splicaris-ing, but the sequences of the corresponding clones from the two lines were identical (data not shown) These results suggest that the CAPER gene is likely not mutated in the R4-7 line Nevertheless,
to test whether any of these cDNAs could alter virus sus-ceptibility, the cDNA inserts from both R4-7 and Rat2 cells were transferred into the expression vector pcDNA3.1/zeo (see Methods) Overexpression of the var-ious CAPER cDNAs from the Rat2 cells in R4-7 cells did not restore virus susceptibility, and overexpression of the
Lack of effect of suppressor cDNAs in wild-type cells
Figure 4
Lack of effect of suppressor cDNAs in wild-type cells Rat2
or a virus-sensitive subclone isolated after mutagenesis
(RC-2) were cotransformed with the indicated DNAs, and the
transformants were pooled and grown into cell populations
The resulting cultures were exposed to N2 virus
(approxi-mately 300 cfu in NIH/3T3 cells) and plated in medium
con-taining G418 All the cultures yielded approximately equal
numbers (~200) of colonies
Sequence alignment of pB1-11 and pC1-2 DNA inserts with
similar sequences from NCBI database
Figure 5
Sequence alignment of pB1-11 and pC1-2 DNA inserts with
similar sequences from NCBI database The insert of pB1-11
(855 bp) is an antisense sequence match to the central
por-tion of the CAPER mRNA [8], and that of pC1-2 (1407 bp) is
a sense sequence match to a portion of the VL30
endog-enous retrovirus-like element [17]
G418r colonies after N2 infection
Rat2 Cells
RC-2
subclone
Alignment of suppressor cDNAs with related sequences
Rat VL30 element
CAPER mRNA
B1-11
A n 5’ cap
C1-2
8 kb
Trang 7corresponding cDNAs from the R4-7 cells did not induce
virus resistance Thus, of the various CAPER expression
constructs, only the original pB1-11 antisense DNA had
biological activity
Discussion
The results here document the development of an
effec-tive procedure for the isolation of cDNAs that allow virus
infection of virus-resistant cells The key feature is the
repeated infection of the parental resistant line with viral
vectors carrying distinct selectable markers, and has
become possible only with the development of a
multi-tude of such markers In this way, rare susceptible cells in
the R4-7 population are enriched by as much as 10 to 100
fold in each round of selection The protocol should allow
the recovery of DNAs that confer susceptibility from any
large library, if present at an abundance of perhaps at least
one in 106 clones The system can only work if a single
DNA is sufficient to enhance virus susceptibility Once cell
lines with restored virus sensitivity were isolated, the recovery of the cDNAs from genomic DNA and the screen-ing for active clones were relatively straightforward The identities of the two sequences in the active cDNAs isolated here were surprising and the mechanisms of action of the two distinct clones remains mysterious Both are highly potent, restoring virus susceptibility essentially
to wild-type levels (figure 2) The time of the block to rep-lication in the R4-7 line that is overcome by these two DNAs is very early after virus entry, before the initiation of reverse transcription by the incoming virus [6] One pos-sibility is that the mutant line fails to uncoat the virions sufficiently to allow deoxyribonucleotides into the core
In this scenario the cDNAs would somehow facilitate the uncoating process or inhibit a block to uncoating HCC1.3 and HCC1.4 are two closely related cDNAs first recovered from a patient with hepatocellular carcinoma [7] The encoded protein was a prominent nuclear autoantigen The deduced amino acid sequences contain
an arginine/serine rich domain and three ribonucleopro-tein consensus sequence domains, often found in RNA
splicing factors; they show weak homology to S pombe
GAR2, a nuclear protein A later report demonstrated that the gene product interacted with the transcriptional acti-vators AP-1 and the estrogen receptors ERa and ERß, and had potent cotransactivation activity; the gene was renamed CAPER, for coactivator of AP-1 and ER [8] The antisense orientation of the pB1-11 cDNA suggests that its mechanism of action might be to lower the level of the endogenous sense mRNAs and the encoded proteins pro-duced from the CAPER gene We were unable to directly assess the level of the mRNAs in the presence of the anti-sense cDNA by Northern blots because the level of expres-sion of the antisense RNA was so much higher than the endogenous mRNA that these transcripts were obscured However, it is possible that the reduction in levels of a protein factor involved in regulation of transcription could elicit profound changes in the patterns of gene expression in the cell We cannot rule out the remote pos-sibility that a cryptic promoter results in some production
of sense mRNA, and a protein fragment with biological activity, from the pB1-11 cDNA
Whatever the mechanism of action of the pB1-11 cDNA,
it is unlikely that the endogenous CAPER gene is the locus
of the original virus resistance mutation in the R4-7 line The levels of the major mRNA are similar in the mutant and the wild-type parent (Fig 6), and sequence analysis of
a variety of cDNAs from parent and mutant lines did not uncover any mutations Further, the overexpression of the CAPER cDNAs from R4-7 did not cause resistance, and the overexpression of the wild-type CAPER cDNA did not sup-press the resistance Rather, the antisense cDNA must
cor-Northern blot analysis of mRNAs in parental Rat2 and
mutant R4-7 cell lines
Figure 6
Northern blot analysis of mRNAs in parental Rat2 and
mutant R4-7 cell lines RNA preparations from the indicated
cells were separated, blotted, and hybridized with a
32P-labeled pB1-11 probe The major mRNA at ~3.0 kb is
indi-cated The position of the 28S and 18S rRNA markers are
indicated on the left
Rat2 R4-7
Northern blot of CAPER mRNAs
CAPER
2.0 kb
5.0 kb
Trang 8rect the phenotype indirectly, likely through effects on
gene expression The localization of the HCC1.3/1.4 or
CAPER protein in the nucleus [7] rather than at the site of
virus arrest also suggests that its mechanism is indirect
Possibly CAPER acts to maintain a program of
cytoplas-mic protein expression that blocks virus infection in the
R4-7 mutant line
The VL30 elements are a very large family of endogenous
virus-like genes found in both mouse and rat genomes
[10-14] The various elements are dispersed and have
sig-nificantly divergent sequences Though gag- and
pol-related sequences are often recognizable, nearly all the
elements are grossly defective, with multiple frameshift
and premature termination mutations interrupting the
open reading frames In addition, the majority of the
elements have suffered deletions of various regions
rela-tive to the longer family members Thus, while many of
the elements are highly transcribed in rodent cell lines,
very few of the transcripts code for protein products of
sig-nificant length However, the VL30 RNAs often contain
recognition elements for packaging into virion particles
encoded by murine leukemia viruses, signals for initiation
of DNA synthesis and strong stop DNA translocation, and
termini recognized by viral integrase proteins, and thus
are competent for transfer by replication-competent
viruses acting as helpers
The sequence of the insert in pC1-2 corresponds to a
por-tion of the retroviral pol gene, specifically the integrase
coding region, but is typical of the VL30s in containing no
long ORF; furthermore, known cis-acting regions needed
for replication are absent We suppose that the RNA itself
may be responsible for the activity, perhaps by binding
some cellular protein The region of the VL30 genome
present in pC1-2 – the 3' portion of the pol gene – is not
known to contain a binding site for any particular protein
The corresponding region of replication-competent
viruses, however, would normally contain the splice
acceptor site for the envelope mRNA Although many
VL30 elements do not contain env genes, and although the
splice acceptor sites are not readily apparent in the pC1-2
sequence, the transcript might be hypothesized to bind
splicing machinery In this scenario, a possible
mecha-nism of action of the clone is for the overexpressed RNA
to bind up a splicing factor or other RNA binding protein,
titering out the free protein and removing it from
solu-tion If this factor were responsible for the viral resistance
of the R4-7, either directly or indirectly, the binding might
relieve that block The mechanism would be surprising
only because the endogenous VL30 RNAs are already so
abundant in Rat2 cells, and they are not able to suppress
the block to infection However, the pC1-2 sequence must
be an unusual element, in that some distinctive aspect of
its sequence must be responsible for its peculiar biological
activity Perhaps identifying proteins that bind to the
pC1-2 transcript would be informative
The mechanism of resistance exhibited by the R4-7 line remains uncertain The block is early but likely not at virus entry: it occurs whether ecotropic envelope, amphotropic envelope, or even the VSV G protein is used for entry [6] Further, the block is unlikely to involve VSV G function, since the cells are susceptible to infection by VSV itself
(J.-W Carroll and M MacDonald, Rockefeller University, unpublished observation) The early block occurs at a similar stage of infection – before reverse transcription –
as the dominant block induced by TRIM5a, a gene respon-sible for retrovirus resistance in primates [5] There are no other indications, however, that the two blocks are related We have observed that the R4-7 cells exhibit a slightly different morphology than the parental Rat2 cells, being somewhat more rounded and more easily detached from the substrate during trypsinization This phenotype could in principle be unrelated to the virus resistance, since the cells were subjected to heavy chemical mutagen-esis before their isolation [6] However, the R4-7 cells expressing both pB1-11 and pC1-2 were restored to a flat-ter morphology, much closer to that of the parental line, suggesting that the two phenotypes may be causally linked If this notion is correct, changes in the cytoskele-ton may be involved in the resistance Further analysis of the R4-7 cells by gene expression profiling may help reveal the basis for its behaviors
Conclusions
The power of genetic selections in mammalian cells for alterations in virus susceptibility is increasing rapidly We believe that selections like the one devised here will be applicable to the isolation of suppressors of other blocks
to infection, including the prototypical Fv1 gene [3], the APOBEC3G cytosine deaminase [4], and the TRIM5a gene [5] The identity of such suppressors may provide impor-tant clues into the mechanism of their action and regulation
Methods
Cell lines, cell culture
The Rat2 cell line is a TK-negative fibroblast line that is highly sensitive to MuLV infection RC-2 is a subclone iso-lated after mutagen exposure but also sensitive to virus, and was used in many experiments as a wild-type control line Lines R3-2 and R4-7 are virus-resistant mutants of Rat2 isolated after exposure to ICR-191 [6] 293T cells are human embryonic kidney cells transformed by adenovi-rus E1 and also expressing SV40 T antigen All these lines were maintained in DMEM with 10% fetal calf serum
Trang 9DNA transformations
A rat kidney cDNA library in the pcDNAI vector [9] was
purchased from Invitrogen (Carlsbad, CA) To increase
the library transfection efficiency and maximize the
integ-rity of the cDNAs, the library was digested in the vector
sequence with the restriction enzyme SfiI and then
reli-gated R4-7 cells in ten 10-cm dishes were cotransformed
with 20 ug of the religated library DNA and 2 ug of
pGK-puro plasmid by calcium phosphate-mediated
transfor-mation Cells expressing transformed DNAs were selected
by growth in culture medium containing 5 ug/ml
puro-mycin Each transformed cell was expected to receive
about 2–10 different cDNAs The cultures were expanded
before the selections for virus susceptibility such that a
pool of 105 cells contained about 2000 distinct
puromy-cin resistant clones Thus, there were about 50 sibling cells
of each transformant in the pools at the time of selection
for virus susceptibility
Retrovirus preparations
neo or MuLV-N2 virus [20]; TK virus, and
Eco-GFP virus [21] were as previously described [6] To
gener-ate Eco-His virus, the Neo resistance gene in the N2 vector
was replaced with His resistance gene, and GP+E86
pack-aging cells [22] were stably transformed with the resulting
vector DNA Recipients were selected with histidinol and
the resistant cells were pooled to generate Eco-His
pro-ducer cells Typical titers of the virus preparations on Rat2
cells were 107 cfu/ml for N2 virus; 2 × 104 for TK virus; 2
× 106 for Eco-His virus; and 105 cfu/ml for Eco-GFP virus
Viral transduction and selection
Selections for virus sensitive cells were performed by
infecting approximately 105 R4-7 cells per 10-cm dish in
each round, with virus titers determined by infection of
Rat2 cells In the first round approximately 104 cfu of N2
virus were applied, and transductants were selected with
800 ug/ml G418 In the second round, approximately 2 ×
103 cfu of Eco-TK virus were used, and transductants were
selected with HAT medium (Gibco) In the third round,
approximately 200 cfu of Eco-His virus were applied, and
transductants were selected with medium containing 1
mg/ml histidinol The multiplicities of all these infections
with the selecting viruses were kept low, at less than 0.1
These low MOIs were required because infection of R4-7
cells at high MOI can override the block, perhaps by
satu-ration of a titratable factor Even at this low MOI, the
pres-ence of many siblings of each transformant implied that
most cDNAs in the pool were tested for inducing virus
susceptibility
Polymerase chain reactions
cDNA inserts from the expression library were recovered
from cell lines by PCR as follows Genomic DNA was
extracted (DNAeasy kit, Qiagen) and subjected to PCR
with primers hybridizing upstream from the CMV pro-moter (sequence 5'-GGGCCAGATATACGCGTT-3') and downstream from the poly(A) addition region (sequence 5'-AATTTGTGATGCTAT-3') of the pcDNAI vector Condi-tions for the PCR were: ten cycles of 94°C for 10 sec, 55°C for 30 sec, and 68°C for 3 min, followed by 20 cycles of the same conditions but with an increase in the polymerase reaction time of 5 sec in each cycle The ampli-fied DNAs were cloned directly into the TOPO vector (Inv-itrogen) and used to transform DH10b bacteria to ampicillin resistance DNAs were isolated from approxi-mately fifty bacterial colonies for each original cell line CAPER cDNAs were prepared from RC-2 mRNA prepara-tions by standard RT-PCR methods using primers span-ning the entire ORF (sequences: 5'- ATATAGCTTAAGGCCACCATGGCAGACGATATTGATAT-3' and 5'-ATATAGGCGGCCGCTCATCGTCTACTT-GGAAC-3'), and cloned into the pcDNA3.1/zeo expres-sion plasmid using AflII and NotI restriction sites
Authors' contributions
GG carried out all the experiments and participated in their design SPG participated in the experimental design and drafted the manuscript Both authors read and approved the final manuscript
Acknowledgements
This work is supported in part by grants to GG from the Ministry of Science and Technology of China (2002AA222041) and CAS Knowledge Innovation Projects (KSCX2-SW-216); and to SPG from the NCI (R01 CA30488) SPG
is an Investigator of the Howard Hughes Medical Institute.
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